Ursolic Acid Attenuates Hepatic Steatosis, Fibrosis, and Insulin Resistance by Modulating the Circadian Rhythm Pathway in Diet-Induced Obese Mice

Abstract

The aim of the current study was to elucidate the effects of long-term supplementation with dietary ursolic acid (UR) on obesity and associated comorbidities by analyzing transcriptional and metabolic responses, focusing on the role of UR in the modulation of the circadian rhythm pathway in particular. C57BL/6J mice were divided into three groups and fed a normal diet, high-fat diet, or high-fat + 0.05% (w/w) UR diet for 16 weeks. Oligonucleotide microarray profiling revealed that UR is an effective regulator of the liver transcriptome, and canonical pathways associated with the "circadian rhythm" and "extracellular matrix (ECM)⁻receptor interactions" were effectively regulated by UR in the liver. UR altered the expression of various clock and clock-controlled genes (CCGs), which may be linked to the improvement of hepatic steatosis and fibrosis via lipid metabolism control and detoxification enhancement. UR reduced excessive reactive oxygen species production, adipokine/cytokine dysregulation, and ECM accumulation in the liver, which also contributed to improve hepatic lipotoxicity and fibrosis. Moreover, UR improved pancreatic islet dysfunction, and suppressed hepatic gluconeogenesis, thereby reducing obesity-associated insulin resistance. Therapeutic approaches targeting hepatic circadian clock and CCGs using UR may ameliorate the deleterious effects of diet-induced obesity and associated complications such as hepatic fibrosis.

Keywords: circadian rhythm; extracellular matrix; fibrosis; liver-specific; ursolic acid.

Figure 1

Effect of ursolic acid (UR) treatment on changes: in body weight (BW) (A); food intake and food efficiency ratio (B); white adipose tissue (WAT) weights (C); brown adipose tissue (BAT) weight (D); WAT morphology (magnification ×200) (E); and plasma lipid levels (F) in C57BL/6J mice fed a high-fat diet (HFD; 20% fat, 1% cholesterol). Data are shown as means ± SEM. Normal diet (ND; AIN-76) vs. HFD; * p < 0.05, ** p <0.01, *** p < 0.001. HFD vs. UR (HFD + 0.05% UR); ^§ p < 0.05, ^§§ p < 0.01, ^§§§ p < 0.001. FER, food efficiency ratio, body weight gain/energy intakes per day; WAT, white adipose tissue; BAT, brown adipose tissue; Total-C, total-cholesterol; HDL-C, HDL-cholesterol; TG, triglyceride; FFA, free fatty acid; HTR, ratio of HDL-C to TC; AI, atherogenic index; Apo, apolipoprotein.

Figure 2

Effect of ursolic acid (UR) treatment on: the liver weight (A); hepatic morphology (magnification ×200) (B); hepatic lipid levels (C); and hepatic lipid-regulating enzyme activities (D) in C57BL/6J mice fed a high-fat diet (HFD). Data are shown as means ± SEM. Normal diet (ND; AIN-76) vs. HFD; * p < 0.05, ** p < 0.01, *** p < 0.001. HFD vs. UR (HFD + 0.05% UR); ^§ p < 0.05, ^§§ p < 0.01, ^§§§ p < 0.001. FAS, fatty acid synthase; PAP, phosphatidate phosphohydrolase; CPT, carnitine palmitoyltransferase; HMGCR, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase; ACAT, acyl-CoA:cholesterol acyltransferase.

Figure 3

Effect of ursolic acid (UR) treatment on: plasma GOT and GPT (A); hepatic mitochondrial H₂O₂ (B); erythrocyte thiobarbituric acid reactive substance (TBARS) (C); plasma paraoxonase (PON) (D); and Masson’s trichrome staining (magnification ×200) (E) in C57BL/6J mice fed a high-fat diet (HFD). Data are shown as means ± SEM. Normal diet (ND; AIN-76) vs. HFD; * p < 0.05, ** p < 0.01, *** p < 0.001. HFD vs. UR (HFD + 0.05% UR); ^§ p < 0.05, ^§§ p < 0.01, ^§§§ p < 0.001. GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; TBARS, thiobarbituric acid reactive substance.

Figure 4

Effect of ursolic acid (UR) treatment on: plasma glucose level (A); insulin level (B); glucagon level (C); homeostasis model assessment-estimated insulin resistance (HOMA-IR) index (D); hepatic glycogen content (E); hepatic glucose-regulating enzyme activities (F); immunohistochemical staining of the pancreatic tissue (G); plasma adipokine (H); and pro-inflammatory cytokine levels (I) in C57BL/6J mice fed a high-fat diet (HFD). Data are shown as means ± SEM. Normal diet (ND; AIN-76) vs. HFD; * p < 0.05, ** p < 0.01, *** p < 0.001. HFD vs. UR (HFD + 0.05% UR); ^§ p < 0.05, ^§§ p < 0.01, ^§§§ p < 0.001. HOMA-IR, homeostasis model assessment for insulin resistance; PEPCK, phosphoenolpyruvate carboxykinase; G6Pase, glucose-6-phosphatase; TNF-α, tumor necrosis factor alpha; MCP-1, monocyte chemoattractant protein-1; IFN-γ, interferon gamma.

Figure 5

Effect of ursolic acid (UR) treatment on: the expression of hepatic clock genes in detoxification (A); clock-controlled genes (CCGs) involved in detoxification (B); lipid and bile acid metabolism (C); transcription patterns of hepatic genes related to lipid metabolism (D); muscular clock genes (E); CCGs (F); and tricarboxylic acid (TCA) cycle-associated genes (G). Symbols in red indicate genes that were up-regulated while those in green denote genes that were down-regulated.

Figure 6

Effect of ursolic acid (UR) treatment on the transcription patterns of hepatic genes related to chemokine/cytokine expression, extracellular matrix (ECM) remodeling, and ECM regulation (A); and ECM–receptor interaction (B). Symbols in red indicate genes that were up-regulated while those in green denote genes that were down-regulated. UR down-regulated collagen, Cd44, Syndecan and Perlecan gene expressions involved in ECM-receptor interaction compared to HFD.